DNA/RNA QUADRUPLEXES: STRUCTURE, STABILITY AND INTERACTIONS

该子项目是利用NIH/NCRR资助的中心赠款提供的资源的许多研究子项目之一。子项目和研究者（PI）可能从另一个NIH来源获得主要资金，因此可以在其他CRISP条目中表示。所列机构为中心机构，不一定为研究者机构。这个项目的重点是不寻常的核酸构象的四链体，建立在鸟嘌呤四。在过去的五年里，这些结构在生物系统中的存在及其功能作用的证据越来越多：例如，四链体结构已被提出在调节端粒酶活性、转录起始、翻译抑制和特异性结合大量蛋白质方面发挥作用。此外，已经描述了四链体作为蛋白质识别的折叠基序、作为除抗病毒和抗癌化疗剂之外的诊断剂的应用。 我们的长期目标是增强对这类核酸的热力学、结构和相互作用的理解，特别是在阳离子结合的特定性质、折叠的RNA四链体的性质及其与DNA四链体的差异以及它们与其他生物学感兴趣的分子的相互作用方面。特别是，我们提出了热力学分析的一系列具有生物学意义的富含鸟嘌呤的序列，无论是在DNA和RNA的形式，这些序列的结构分析，开发的方法来评估四链体形式的分子和催化DNA构建体及其与氯化血红素的相互作用的详细调查。 在这一目标中采用的实验技术包括UV/CD光谱，等温滴定量热法，NMR，荧光和平衡沉降。此外，质谱法将用于在旨在表征生物条件下人类端粒结构的实验中确认连接产物。这种方法的组合将提供非常重要的核酸种类的构象和相互作用性质的详细检查。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. This project focuses on the unusual nucleic acid conformation of quadruplexes, built upon the guanine quartet. Evidence for the presence of these structures in biological systems, and their functional roles, has been mounting over the past five years: for example, quadruplex structures have been proposed to play a role in regulating telomerase activity, in initiation of transcription, in translational repression, and in binding specifically to a substantial number of proteins. Furthermore, applications have been described for quadruplexes as a folding motif for protein recognition, as diagnostic agents in addition to antiviral and anticancer chemotherapeutic agents. Our long term goal is an enhanced understanding of the thermodynamics, structure and interactions of this class of nucleic acids, in particular in terms of the specific nature of cation binding, the properties of folded RNA quadruplexes and their differences with respect to DNA quadruplexes, and their interactions with other molecules of biological interest. In particular, we propose thermodynamic analyses of a series of biologically significant guanine-rich sequences, both in DNA and RNA forms, structural analyses of these sequences, development of methods to assess the molecularity of quadruplex forms and a detailed investigation of a catalytic DNA construct and its interactions with hemin. Experimental techniques to be employed in this goal include UV/CD spectroscopy, isothermal titration calorimetry, NMR, and fluorescence, and equilibrium sedimentation. Additionally, mass spectrometry will be used to confirm ligation products in experiments aimed at characterizing the human telomere structure under biological conditions. This combination of approaches will afford a detailed examination of the conformation and interaction properties of the very important nucleic acid species.